Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates

L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.     

Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.     

Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports

Glucoamylase from four different companies was studied: three had similar stability (half-life at 50°C about 140 hr); the fourth was less stable (half-life at 50°C about 20 hr). The immobilized enzymes were all less stable than their soluble counterparts: immobilized enzyme stability depended on the soluble enzyme used, the support, and method of immobilization. Thus enzyme bound to Enzacryl-TIO was less stable than enzyme bound to hornblende (metal-link method); this, in turn, was less stable than enzyme bound to hornblende by a silane–glutaraldehyde process. Bound enzyme stability was also improved by the presence of substrate or product (starch maltose or glucose). After 110 hr at 50°C in the presence of maltose (10% (w/v)) one preparation (a more stable soluble enzyme boul1d to hornblende by a silane–glutaraldehyde process) retained over 95% of its activity: activity loss was too low to permit the estimation of a half-life.     

Effect of immobilization on stability and properties of N-acetyl-β-d-hexosaminidase from Turbo cornutus

A mixture of glycosidases from the liver of the gastropod Turbo cornutus was co-immobilized with bovine serum albumin and glutaraldehyde, and then cast as membranes. The properties of immobilized N-acetyl-β-d-hexosaminidase were studied. The recovery of N-acetyl-β-d-hexosaminidase after immobilization was unaffected by increasing the concentration of glutaraldehyde, but was decreased by increasing the bovine serum albumin concentration. The immobilized enzyme showed enhanced resistance towards proteolytic and thermal inactivation. While the pH optimum for the soluble enzyme was 4.0, a bimodal pH curve with optima at 3.4 and 5.0 was observed after insolubilization. This bimodality was abolished when the immobilized enzyme was assayed in the presence of M NaCl. The K_m values, for p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside, of the immobilized isoenzymes of N-acetyl-β-d-hexosaminidase were larger than those of their soluble counterparts. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage for at least 50 days at 4°.     

Proteins adsorbed to a hydrophobic surface used for determination of proteolytic activity

Wettability of a thin layer of protein adsorbed to a hydrophobic surface is reduced after proteolytic digestion. Reduced wettability is demonstrated by condensation of water vapour on the surface. The condensation patterns of enzyme-treated and untreated protein layers give different light-scattering properties which can be observed by the naked eye. Based on these principles, a new simple and inexpensive method, thin layer enzyme assay (TEA), for determination of proteolytic activity, was developed. Fibrinogen, gammaglobulin (IgG), bovine serum albumin (BSA), haemoglobin, ovalbumin and gelatin were used as substrates. The proteolytic activity in 1 ng trypsin (EC 3.4.21.4) and in 1 ng pronase (EC 3.4.24.4) was reproducibly detected.     

Investigation of Digestion and Absorption of a Dietary Protein by Using Its Antigenicity as an Index: Gastrointestinal Digestion of Ovalbumin

An attempt was made to use the disappearance of ovalbumin antigen as an index for the analysis of the process of digestion of ovalbumin in vitro and in vivo. The antigenic determinants of ovalbumin were highly sensitive to conformational changes in the protein. Mild heat-treatment and acid-denaturation abolished spontaneously all the reactivities of antigenic determinants. Experiments on protease digestion in vitro showed that native ovalbumin was hardly digested by pepsin at pH 2.7 or by pancreatin at pH 8.0, while acid- or heat-denatured ovalbumin was more easily digested by these proteases. The results suggested that the denaturation of ovalbumin was a rate-limiting step in its digestion, and the disappearance of ovalbumin antigen was a useful index of the initial process of digestion which preceded the proteolytic hydrolysis.

At 1 hr after the administration of an ovalbumin diet to rats, approximately 20% of the ovalbumin antigen administered was detected in the whole contents of the gastrointestinal tract. In the small intestine, the amount of intact ovalbumin comprised about half of the total protein of the intestinal contents. These results suggested that in the stomach and small intestine, denaturation of ovalbumin was a significant rate-limiting step in its digestion process. But after 2hr, little ovalbumin antigen was detected in the small intestine. This disappearance of the antigen from the small intestine cannot be well explained by the in vitro data.     

PURIFICATION AND PROPERTIES OF DETERGENT-COMPATIBLE EXTRACELLULAR ALKALINE PROTEASE FROM Scopulariopsis spp.

A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0–12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co²⁺ and Mn²⁺ at 10 mM but was unaffected by Ba²⁺, Mg²⁺, Cu²⁺, Na⁺, K⁺, and Fe²⁺. Ca²⁺ and Fe³⁺ moderately reduced the activity (～18%); however, a reduction of about 40% was seen for Zn²⁺ and Hg²⁺. The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H₂O₂ and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (?20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel–enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity, stability in the presence of surfactants and oxidizing and bleaching agents, and excellent compatibility with detergents clearly suggested the use of the enzyme in detergent formulations.     

The reactions between formaldehyde,glutaraldehyde and osmium tetroxide,and their fixation effects on bovine serum albumin and on tissue blocks

Summary The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.     

Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis

In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

Extracellular proteolytic enzyme activity ofHistoplasma capsulatum var.duboisii

Histoplasma capsulatum var.duboisii is the etiological agent of African histoplasmosis, an important deep mycosis in West Africa. Not much is known about the physiological properties of this fungus. This communication reports on the extracellular proteolytic enzyme activity of this fungus. Five isolates of this fungus tested hydrolyzed azocasein and bovine serum albumin at pH 6.8 and 8.0. Assay of the crude enzyme showed that proteolytic activity increased with age and peaked on the 10th day and then again on the 13th day for the yeast form, and on the 11th day of growth for the mycelial form. The optimum temperature and pH for maximum enzyme activity were 35 °C and 6.8 respectively. The proteinase activity was more pronounced with the yeast form than with the mycelial form. The action of enzyme inhibitors suggested the presence of an aspartyl proteinase.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Polymerization of Several Proteins by Ca2+-Independent Transglutaminase Derived from Microorganisms

α_s1-Casein and soybean globulins were polymerized and gelatinized by Ca²⁺-independent transglutaminase that was isolated from the culture filtrate of a microorganism thought to belong to Streptoverticillium sp. of actinomycetes. This enzyme polymerized such albumins as bovine serum albumin, human serum albumin and conalbumin in the presence of dithiothreitol. Rabbit myosin was polymerized by the present emzyme but actin was not. An RP-HPLC analysis after enzymic digestion of the polymerized a_sl -casein showed existence of the £-(y-Glu)Lys bond. Thus, it was confirmed that the polymerization was formed by a catalytic reaction of the transglutaminase.     

Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.     

Preparation and characterization of amino and carboxyl functionalized core-shell Fe3O4/SiO2 for L-asparaginase immobilization: A comparison study

Abstract

Magnetic nanoparticles are well known as facile and effective support for enzyme immobilization since they have a high surface area, large surface-to-volume ratio, easy separation, a fast and high enzyme loading. This study aims to provide insights on whether acidic or basic modified particles are more effective for L-asparaginase (ASNase) immobilization. Therefore, amino (Fe₃O₄/SiO₂/NH₂) and carboxyl-functionalized (Fe₃O₄/SiO₂/COOH) particles were prepared. The functional groups, crystalline structure, magnetic properties, morphology, chemical composition and thermal behaviour of the prepared modified nanoparticles were examined via Fourier-transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), vibrating-sample magnetometer (VSM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDAX). Under the optimum conditions, the immobilized enzymes were more stable within a certain range of temperatures and pH values in comparison to free enzyme. On the other hand, the immobilized enzymes showed greater stability after incubation for 3?h at 50?°C. The free enzyme maintained only 30% of its initial activity for 4?weeks at 4?°C, while Fe₃O₄/SiO₂/NH₂/ASNase and Fe₃O₄/SiO₂/COOH/ASNase retained more than 78.9% and 56.5% of initial activities under the same conditions, respectively. Moreover, Fe₃O₄/SiO₂/NH₂/ASNase (77.2%) and Fe₃O₄/SiO₂/COOH/ASNase (57.4%) displayed excellent operational stability after 17 repeated cycles. These findings suggested that the Fe₃O₄/SiO₂/NH₂ and Fe₃O₄/SiO₂/COOH may be utilized as efficient and sustainable supports to developed immobilized ASNase in several biotechnological applications.     

KINETIC STUDIES OF L-ASPARAGINASE FROM Penicillium digitatum

L-Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and other related malignancies. Its further use includes reduction of asparagine concentration in food products, which may lead to formation of acrylamide. Currently bacterial asparaginase is produced at industrial scale, but the enzyme isolated from bacterial origin is often associated with adverse reactions. These side effects require development of asparaginase from alternative sources. In the present study, Penicillium digitatum was explored for the production of extracellular L-asparaginase using modified Czapek–Dox media. The enzyme was purified about 60.95-fold and then kinetic study showed that the Km value of the enzyme was 1 × 10^?5 M. The optimum pH and temperature for the enzyme were 7.0 and 30°C, respectively. The optimum incubation period for L-asparaginase was 15 min. This work concludes that this enzyme can be a suitable candidate due to its strong kinetic properties, and further research can usher into development of asparaginase formulation from fungal origin with less adverse effects.     

Preparation and properties of co-reticulated invertase supported by an ultrafiltration membrane.

Yeast invertase was co-reticulated with glutaraldehyde to bovine serum albumin to give a soluble bound enzyme that was immobilized as a tightly adhering layer on the active surface of an ultrafiltration membrane. The Michaelis constant and stability of this immobilized-enzyme system are compared with those of the enzyme either in the native form or immobilized as a dynamically formed gel layer on an ultrafiltration membrane, as previously described by us [Drioli, Gianfreda, Palescandolo & Scardi (1975) Biotechnol, Bioeng, 17, 1365-1367].     

Flow Injection Analysis of Lactose in Milk Using a Chemically Modified Lactose Electrode

β-Galactosidase and glucose oxidase were immobilized with bovine serum albumin using glutaraldehyde on to a glassy carbon electrode silanized with 3-aminopropyltriethoxysilane. The laboratory-constructed lactose electrode was used for flow injection analysis to determine the lactose content in milk. Electrochemical interference could be detected by a non-enzymatic electrode (W₂) and the current was subtracted from that of the enzymatic electrode (W₁), providing an accurate measurement of the hydrogen peroxide that was enzymatically generated. The peak current was linearly related to the lactose concentration in the range 10^?4~ 1.5 × 10^?3 M (original concentration) and 40 samples/hr could be analyzed. The relative standard deviation for 10 assays was less than 2%. The proposed method was compared with the chloramine T method and the values determined by both methods were in good agreement.     

Purification and partial characterization of an elastinolytic proteinase from Aspergillus flavus culture filtrates

 A 23-kDa protein with elastinolytic activity was purified from Aspergillus flavus (NRRL 18543) culture filtrates by gel-filtration chromatography. Severe inhibition of the elastinolytic activity by 1,10-phenanthrolene (5 mM) and EDTA (0.8 mM) indicated that the protein belongs to the metallo class of proteases. The isoelectric point was 9.0. Natural substrates susceptible to cleavage by this protease, in addition to elastin, included cottonseed storage protein, collagen, ovalbumin and bovine serum albumin. The 23-kDa protein was thermostable to 70°C and retained its elastinolytic activity in concentrated form at 4°C for 6 months. Elastinolytic activity was initially secreted into the culture medium as a 35-kDa protein, which was subsequently converted to a 23-kDa protein, presumably through autolysis. This putative proteolytic degradation product appears to be identical to the 23-kDa protein recovered from the gel-filtration column. The 23-kDa protease may confer selective advantage to the fungus in the extracellular environment because of its temperature and pH stability and wide range of potential natural protein substrates. Received: 24 October 1995/Received last revision: 27 March 1996/Accepted: 30 March 1996